During development of the nervous system the response of growing axons to their environment is critical to the formation of the complex wiring pattern between neurons. Growth and guidance factors combined with extracellular matrices influence the speed and direction of axonal growth. Although much progress has been made in identifying the factors that influence axonal growth, as well as how axons respond to these factors individually, much less is known about how axons behave in response to the combined effects of multiple factors. As a complementary approach to present in vivo molecular imaging approaches, we propose to develop an in vitro environment that potentially mimics some of the complexity found in vivo, in particular the development of the anterior visual pathway. In this system, the axon trajectories are simple, multiple relevant guidance molecules have been identified already (many tested with explants in vitro), and a common cause of blindness (Optic Nerve Hypoplasia) is associated with defects in this process. Additionally, the patterns of guidance molecules found on the flat anatomy of the retina are ideally suited to mimicking by micropatterning and microfluidics techniques. This mimicry will be accomplished by combining microfluidics patterning of diffusible gradients and laser patterning of substrate-bound axon pathfinding cues, including axon guidance factors and extracellular matrix molecules. As a source of highly homogeneous cell populations, we will isolate mouse retinal ganglion cells (RGCs), a cell type that responds to Netrin-1 gradients. For experiments designed to maximize the integrity of the cells (isolation procedures are damaging to cells), we will use retinal explants and we will microfluidically isolate the axons from their somas. RGCs (or their axons) will be exposed to various soluble factors that have previously been shown to affect their axon growth in vivo. The new microfluidic systems will allow us to test the combinatorial effects of multiple factors on the direction and speed of axonal growth of RGCs. These experiments will allow us to quantitatively examine the basic principles that govern axon pathfinding in the development of the anterior visual pathway. This information will help to better understand the basis of developmental defects in axon growth that alter the organization and function of the nervous system.